Hydroxylation of methyl groups in aromatic heterocycles by microorganisms

Abstract

A process using microorganisms which contain genes, which form an active xylene monooxygenase, which form no effective, chromosomally or plasmid-coded alcohol hydrogenase, and which are, thus, capable of hydroxylating methyl groups on aromatic 5- or 6-atom heterocycles to the corresponding hydroxymethyl derivatives, for the production of hydroxymethylated 5- or 6-atom heterocycles.

What is claimed is: 
     
       1. Microbiological process comprising hydroxylating the methyl group or methyl groups in a methylated 5- or 6-atom aromatic heterocycle, the reaction being performed with microorganisms which: (a) contain the genes of a Pseudomonas TOL plasmid, which form an active xylene monooxygenase, and   (b) form no effective chromosomally or plasmid-coded alcohol dehydrogenase, and, thus, are capable of hydroxylating the methyl group or methyl groups of the methylates 5- or 6-atom aromatic heterocycle to the corresponding hydroxymethyl derivative, the heterocycle being used as a substrate for the reaction and having no substituents on the carbon atom adjacent to the methyl group to be hydroxylated, and the hydroxymethyl derivative not being further catabolized.   
     
     
       2. Process according to claim 1 wherein the reaction is performed with a microorganism in which the genes come from a Pseudomonas TOL plasmid which form an effective xylene monooxygenase and are characterized by the restriction map in the FIGURE. 
     
     
       3. Process according to claim 2 wherein the reaction is performed with a microorganism which belongs to a genus selected from the group consisting of Pseudomonas, Acinetobacter, Rhizobium, Agrobacterium and Escherichia. 
     
     
       4. Process according to claim 3 wherein the reaction is performed with the microorganism of species Escherichia coli K12*, transformed with hybrid plasmid pGSH2836 (DSM no. 6154), or a descendant thereof or a mutant thereof. 
     
     
       5. Process according to claim 3 wherein the reaction is performed with the microorganism of species Escherichia coli K12* transformed with hybrid plasmid pL04 (DSM No. 6153), or a descendant thereof or a mutant thereof. 
     
     
       6. Process according to claim 3 wherein the reaction is performed with the microorganism of species Pseudomonas putida JD7 transformed with hybrid plasmid pL05 (DSM no. 6152), or a descendant thereof or a mutant thereof. 
     
     
       7. Process according to claim 6 wherein the reaction is performed with a methylated 5- or 6-atom heterocycle, which contains one or more heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. 
     
     
       8. Process according to claim 7 wherein the reaction is performed either with single or continuous addition of substrate so that the substrate concentration in the culture medium does not exceed 20 percent (w/v) or (v/v). 
     
     
       9. Process according to claim 8 wherein the reaction is performed at a pH of 4 to 11 and at a temperature of 15° to 50° C. 
     
     
       10. Process according to claim 1 wherein the reaction is performed with a microorganism, which contains a natural TOL plasmid, in which the gene, which is the code for an effective alcohol dehydrogenase, is removed or deactivated. 
     
     
       11. Process according to claim 1 wherein the reaction is performed with a microorganism which belongs to a genus selected from the group consisting of Pseudomonas, Acinetobacter, Rhizobium, Agrobacterium and Escherichia. 
     
     
       12. Process according to claim 1 wherein the reaction is performed with the microorganism of species Escherichia coli K12*, transformed with hybrid plasmid pGSH2836 (DSM no. 6154), or a descendant thereof or a mutant thereof. 
     
     
       13. Process according to claim 1 wherein the reaction is performed with the microorganism of species Escherichia coli K12* transformed with hybrid plasmid pL04 (DSM no. 6153) or a descendant thereof or a mutant thereof. 
     
     
       14. Process according to claim 1 wherein the reaction is performed with the microorganism of species Pseudomonas putida JD7 transformed with hybrid plasmid pL05 (DSM no. 6152), or a descendant thereof or a mutant thereof. 
     
     
       15. Process according to claim 1 wherein the reaction is performed with a methylated 5- or 6-atom heterocycle, which contains one or more heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. 
     
     
       16. Process according to claim 1 wherein the reaction is performed with either single or continuous addition of substrate so that the substrate concentration in the culture medium does not exceed 20 percent (w/v) or (v/v). 
     
     
       17. Process according to claim 1 wherein the reaction is performed at a pH of 4 to 11 and at a temperature of 15° to 50° C.